Andrew R. Collins

Oxidative DNA damage, antioxidants, and cancer.

Oxidised bases, such as 8‐oxo‐guanine, occur in cellular DNA as a result of attack by oxygen free radicals. The cancer‐protective effect of vegetables and fruit is attributed to the ability of antioxidants in them to scavenge free radicals, preventing DNA damage and subsequent mutation. Antioxidant supplements (e.g., β‐carotene, vitamin C) increase the resistance of lymphocytes to oxidative damage, and a negative correlation is seen between antioxidant concentrations in tissues and oxidised bases in DNA. Large‐scale intervention trials with β‐carotene have, however, led to increases in cancer. Recent measurements of the frequency of oxidised DNA bases indicate that earlier estimates were greatly exaggerated; there may be only a few thousand 8‐oxo‐guanines per cell. Convincing evidence for mutations resulting from oxidative damage, in tumours or cultured cells, is lacking. It seems that efficient antioxidant defences together with DNA repair maintain a steady‐state level of damage representing minimal risk to cell or organism. BioEssays 21:238–246, 1999.

Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage

We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2′-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within ±40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, 32P-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision and detection of dose response, cannot at present be recommended.

UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts.

Rodent UV‐sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS‐B and CS‐A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild‐type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6‐4) photoproduct removal. ERCC6, the product of the gene defective in CS‐B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockaynesyndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40–50% of dimers being removed in both wild‐type and group 6 mutants in 5 hours following 0.1 Jm‐2 of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS‐A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells. Environ. Mol. Mutagen. 29:152–160, 1997.

A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity

A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.

How the 1932 and 1947 mental surveys of Aberdeen schoolchildren provide a framework to explore the childhood origins of late onset disease and disability.

OBJECTIVES To describe the discovery and development of the Aberdeen 1921 and 1936 birth cohort studies. STUDY DESIGN The Aberdeen birth cohort studies were started in 1998 when the Scottish Mental Survey archives of the Scottish Council for Research in Education were re-discovered and permissions granted to follow-up survivors born in 1921 or 1936 and then aged about 77 or 64 years and who had entered (or were about to enter) the age of greatest risk for Alzheimers disease (AD). MAIN OUTCOME MEASURES Sources of attrition from the study, exposures to childhood adversity, nutritional, genetic and life style factors of possible relevance to extent of age-related cognitive decline and the timing of onset of dementia. RESULTS By 2010, the feasibility of following up more than 75% of Scottish Mental Survey survivors living in the Aberdeen area without dementia was well-established, dementia ascertainment to age about 88 years was completed in the 1921 birth cohort and was underway in the 1936 born cohort. CONCLUSION These databases are available to other bone fide research groups wishing to test specific hypotheses that may either replicate their own findings or make best use of the data collected in the Aberdeen studies.

Phenotypic heterogeneity in nucleotide excision repair mutants of rodent complementation groups 1 and 4

Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.

Deleterious consequences of antioxidant supplementation on lifespan in a wild-derived mammal

While oxidative damage owing to reactive oxygen species (ROS) often increases with advancing age and is associated with many age-related diseases, its causative role in ageing is controversial. In particular, studies that have attempted to modulate ROS-induced damage, either upwards or downwards, using antioxidant or genetic approaches, generally do not show a predictable effect on lifespan. Here, we investigated whether dietary supplementation with either vitamin E (α-tocopherol) or vitamin C (ascorbic acid) affected oxidative damage and lifespan in short-tailed field voles, Microtus agrestis. We predicted that antioxidant supplementation would reduce ROS-induced oxidative damage and increase lifespan relative to unsupplemented controls. Antioxidant supplementation for nine months reduced hepatic lipid peroxidation, but DNA oxidative damage to hepatocytes and lymphocytes was unaffected. Surprisingly, antioxidant supplementation significantly shortened lifespan in voles maintained under both cold (7 ± 2°C) and warm (22 ± 2°C) conditions. These data further question the predictions of free-radical theory of ageing and critically, given our previous research in mice, indicate that similar levels of antioxidants can induce widely different interspecific effects on lifespan.

The mode of action of 1-β-d-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.

Aberrant DNA repair and enhanced mutagenesis following mutagen treatment of Chinese hamster Ade−C cells in a state of purine deprivation

Ade-C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.

Deoxyribonucleotide Pools, DNA Repair and Mutagenesis

The DNA resynthesis step of excision repair makes only a small demand on the pool of DNA precursors in the cell. However, DNA repair is sensitive to drugs that act via the dNTP pool; hydroxyurea, which depletes certain of the dNTPs by inhibiting ribonucleotide reductase, slows the resynthesis step and causes incomplete repair sites to accumulate as easily measurable DNA breaks. The dependence of DNA repair on nucleotide synthesis is examined in a mutant cell line, Ade- C., a purine auxotroph. When deprived of hypoxanthine as a purine source, Ade-C cells cease DNA replication, and although DNA repair continues, it is abnormal. Purine starvation increases both the cytotoxicity and the mutagenicity of ultraviolet light. We find 4–5 times more forward or back mutations in Ade-C cells irradiated in the purine-starved state compared with irradiated but unstarved cells.